With respect to peptides and proteins collected from nature, the identification of their amino acid sequences are essential information to make a study of the biological properties and functions of the peptides and proteins in question. Currently, the full-length amino acid sequences for peptides and proteins are determined as deduced amino acid sequences, based on corresponding gene information thereof, for instance, nucleotide sequences of the genomic genes or c-DNAs produced from m-RNAs which encode their peptides. However, in identifying the genomic genes or the c-DNAs produced from m-RNAs which encode these peptides, the knowledge of partial amino acid sequences of the peptides is still required.
It is generally considered that, as the knowledge of the partial amino acid sequences of peptide, the N-terminal amino acid sequence and C-terminal amino acid sequence of peptide are particularly useful. Specifically, for example, in selecting a c-DNA which encodes an aimed peptide from a c-DNA library prepared from a large number of m-RNAs, if the N-terminal amino acid sequence and C-terminal amino acid sequence thereof are known, the aimed c-DNA can be selected by using nucleic acid probes that are produced based on said amino acid sequences of the two termini. Alternatively, the aimed c-DNA can be amplified selectively by applying PCR with use of oligonucleotide primers that are produced based on the amino acid sequences of the two termini.
As the method for analyzing the N-terminal amino acid sequence of a peptide, there has been conventionally used a method of subjecting a pure peptide sample obtained by isolation and purification to Edman degradation to successively degrade the N-terminal amino acids therefrom and identify the resulting amino acid derivatives. Meanwhile, as the method for analyzing the C-terminal amino acid sequence of a peptide, there has been proposed a method comprising steps of releasing the C-terminal amino acids successively from such a pure peptide sample by means of chemical technique and identifying the C-terminal amino acids released thereby, based on the molecular weight differences between the original peptide and truncated peptides that are obtained as reaction products therefrom. As the process for releasing the C-terminal amino acids successively by means of chemical technique, there is proposed, for example, a process comprising steps of allowing a vapor generated from a high concentration aqueous solution of pentafluoropropanoic acid (CF3CF2COOH) or a high concentration aqueous solution of heptafluorobutanoic acid (CF3CF2CF2COOH), to act on a dried pure peptide sample under heating up condition of at 90° C., and thereby carrying out selective hydrolysis of the C-terminal amino acids, which is enhanced by said perfluoroalkanoic acid [Tsugita, A. et al., Eur. J. Biochem. 206, 691-696 (1992)]. In addition, there is also proposed a process using, in place of said high concentration aqueous solution of a perfluoroalkanoic acid, a solution of pentafluoropropanoic acid anhydride [(CF3CF2CO)2O] in acetonitrile or a solution of heptafluorobutanoic acid anhydride [(CF3CF2CF2CO)2O] in acetonitrile, which process comprises steps of allowing a vapor generated from the solution, to act on a dried peptide under cooling down condition, for example, at −18° C., and thereby conducting selective release of the C-terminal amino acids, which is forced by said perfluoroalkanoic acid anhydride [Tsugita, A. et al., Chem. Lett. 1992, 235-238; Takamoto, K. et al., Eur. J. Biochem. 228, 362-372 (1995)].
In said method for selectively releasing the C-terminal amino acids by allowing a perfluoroalkanoic acid or a perfluoroalkanoic acid anhydride, which are supplied in vapor phase as a vapor thereof, to act on a dried pure peptide sample, it has been reported that an oxazolone ring structure is once formed from the C-terminal amino acids, as a reaction intermediate, through a dehydration reaction shown by the following reaction scheme (I):
and then, the perfluoroalkanoic acid acts on the oxazolone ring to give rise to a reaction shown by the following reaction scheme (II):
as a result, reaction of selectively releasing the C-terminal amino acids therefrom is achieved.
As the above reaction of selectively releasing the C-terminal amino acid proceeds successively, there is obtained, at a timing when a given treatment time has passed, a mixture comprising a series of reaction products in which one to ten odd amino acid residues have been removed from the C-terminus of the original peptide, respectively. This mixture comprising a series of reaction products is subjected to mass spectrometry to measure the masses of the ion species derived from the reaction products, whereby can be obtained a series of peaks exhibiting the mass differences, which reflect the C-terminal amino acid sequence. Specifically explaining, the individual reaction products are formed in reaction of successively releasing the C-terminal amino acids from the original peptide; hence, for example, a set of reaction products including several members in series, where up to several amino acid residues have been removed from the original peptide, are subjected to mass spectrometry and, thereby, the masses of corresponding ion species thereto can be analyzed collectively, which enables determination of C-terminal amino acid sequence of such several amino acid residues at one time.
Incidentally, for example, the information of C-terminal amino acid sequence used in production of nucleic acid probe or primer may originally be, in terms of the nucleotide sequence which codes such amino acid sequence, about 18 to 24 bases and accordingly about 6 to 8 amino acids. The identification of C-terminal amino acid sequence of up to ten odd amino acid residues is required only in very rare cases. Therefore, the above methods for preparation of treated sample comprising a series of reaction products, in which all the removals extending up to 10 odd amino acid residues are included, by the reaction of releasing the C-terminal amino acids from the dried peptide, where a vapor of a perfluoroalkanoic acid or a perfluoroalkanoic acid anhydride are supplied in vapor phase and allowed to act thereon, are suitable for the above-mentioned purposes.